Signaling by TGF-beta is regulated at multiple levels in order to generate specificity and to finely tune these signals. One of these regulatory mechanisms is the ubiquitin-proteasome pathway, which controls the level and/or activity of Smad proteins through degradation, thereby modulating cellular responsiveness to TGF-beta ligands. We, and others, have previously identified two Smad ubiquitin regulatory factors (Smurfs) of the HECT domain-containing ubiquitin ligase family and shown that Smurf1 and Smurf2 have the ability to interact directly with Smad1 and Smad5 of the BMP pathway and mediate their degradation. To address the physiological significance of Smurfs in TGF-beta signaling, we have generated mice lacking either Smurf1 or Smurf2, and reported that Smurf1-deficient mice are perinatally normal but exhibit an age-dependent increase of bone mass due to enhanced osteoblast activity and increased responsiveness to BMP. Surprisingly, this skeletal abnormality is not caused by alteration in Smad-mediated TGF-beta or BMP signaling. Instead, loss of Smurf1 results in accumulation of phosphorylated MEKK2 in osteoblasts and activation of its downstream JNK signaling cascade. Our results reveal a novel function of Smurf1 in the regulation of osteoblast physiology and bone homeostasis, and provide an interesting example for the importance of the mitogen-activated protein kinase (MAPK) signaling pathway in shaping specific biological response to the TGF-beta family of cytokines. Currently, we are characterizing the phenotypes of Smurf2 deficient mice and Smurf1/Smurf2 double deficient mice to investigate how Smurf-mediated ubiquitination affects cell growth, tissue differentiation and other biological processes regulated by the TGF-beta family of ligands.Although Smads are involved in most actions of the TGF-beta superfamily, activated TGF-beta receptors also transduce signals through other intracellular signaling pathways, especially those mediated by MAP kinases. The second area of research of my group focuses on the specific mechanism by which TGF-beta receptors activate MAP kinases independent of Smads, and the biological significance of this non-Smad dependent pathway in TGF-beta signaling. Toward this goal, we found that TRAF6 is specifically required for the Smad-independent activation of JNK and p38 and its carboxyl TRAF homology domain physically interacts with TGF-beta receptors. TGF-beta induces K63-linked ubiquitination of TRAF6, and promotes association between TRAF6 and TAK1. Our results indicate that TGF-beta activates JNK and p38 through a mechanism similar to that operating in the interleukin-1beta/Toll-like receptor pathway. Currently, we seek to expand this findings to characterize molecular mechanisms of the Smad-independent pathways by identifying proteins that are specifically associated with TGF-beta type I receptor and characterizing their functions. The third direction of my group focuses on the effect of aberrant Smad signaling in tumorigenesis. We have generated different lines of transgenic mice carrying either wild type, or dominant negative or Smad3 under the control of a tetracycline-repressible promoter (tet-off). We crossed these mice to LAP-tTA mice, which allow tetracycline-regulated expression of tetracycline-transactivating protein (tTA) specifically in hepatocytes, to express Smad3 and its variants in liver. We find that elevated Smad3 expression protects liver from chemically induced carcinogenesis due to a heightened hepatic response to apoptotic stimuli. We plan to continue using this model to further explore the role of Smad3 in late stages of liver tumor progression and metastasis. In addition, we are also interested in how Smad signaling converges with other pathways and what kind of roles these cross-talks play in controlling TGF-beta-regulated gene transcription, cell proliferation, differenciation, apoptosis and tumor progression.